Long term potentiation (LTP) and long term depression (LTD) in developing and mature nervous systems require the release of calcium from smooth ER located at the synapse. Myosin V is postulated to play an important role in this process by transporting ER to the synapse where repetitive electrical activity triggers calcium release. Genetic studies with dilute (MyoVa null) mice have shown that mutations in myosin V lead to neurological defects including opisthotonos and ataxia. The neurological disorder in humans known as Griscelli Syndrome has been linked to mutations in the human gene for myosin V.In this research project we plan to provide additional evidence that transport of ER in the actin-rich regions of neurons is driven by myosin V. We will use the squid nervous system as a model for these studies. ER vesicles in the squid giant axon retain the ability to move on actin filaments in vitro and we have shown that myosin V is the vesicle motor. Our lab was the first to show that these vesicles move on both microtubules and actin filaments, an observation that led to the 'dual transport' model. Our principal goal is to determine the mechanism by which vesicle transport on microtubules and actin filaments is coordinated and regulated. We plan to test the hypothesis that myosin V and kinesin form a complex on vesicles through tail-tail interactions and that feedback between the proteins facilities the transition of vesicles from microtubules to actin filaments.The overall goal of this research project is to isolate, identify and characterize the myosin-V/kinesin motor complex. The first specific aim is to isolate the complex and to determine the proteins that link the complex to the surface of vesicles. The second specific aim of this project is to establish that the tail domains of myosin V and kinesin interact directly when the two motors form a complex on vesicles. The third specific aim is to determine if binding to cargo (vesicles) activates the motors and whether feedback between the tails of myosin V and kinesin alters motor activity. Myosin V complexes and vesicle fractions will be isolated from squid brain. Biochemical techniques including chemical cross-linking, affinity chromatography and immunoprecipitation will be used to identify novel proteins in the hetero-motor complex. Novel proteins will be cloned and sequenced. Co-sedimentation assays and FRET microscopy will be used to determine if feedback between the two motors in the multi-protein complex alters motor activity. These studies will advance our understanding of the mechanism by which long-range movement of vesicles on microtubules and short-range movement on actin filaments are coordinated.